Materials
P. igniarius (CGMCC 5.95) was purchased from the China Microbial Preservation Center (Beijing, China) and was cultured in modified Martin medium (MMM), including 5 g/L peptone, 1 g/L K2HPO3, 2 g/L yeast extract, 20 g/L glucose, 20 g/L agar, pH 6.2-6.5. The culture medium was sterilized by autoclave at 121 ℃ for 30 min. The strain was stored at 4 ℃.
HPLC detection of metabolites by precursor feeding
P. igniarius was cultured in MMM medium for seven days at 28 ℃. An agar patch (1 cm × 1 cm) was added into a 250 mL conical bottle containing 150 mL liquid medium (the above MMM without agar). The culture conditions were 28 °C, 180 rpm. Then, 15 days later, the TL, which was dissolved in double distilled water, was added into the fermentation liquor at a final concentration of ~0.1 mg/mL. The co-culture was then allowed to grow for another 9 days under the same conditions. Samples (2.0 mL) were taken at 1, 3, 5, 7, and 9 days after the addition of TL, and were extracted by EtOAc (3 × 2.0 mL). The organic layer was removed by volatilization and the residue was dissolved in 2 mL methanol for HPLC detection. HPLC was conducted using Agilent 1220 Infinity II equipment with Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm) as the separation column. Two-phase gradient elution methods were used in this work including 0.2% formic acid (an aqueous solution) as phase A, and MeOH as phase B. The MeOH fraction in phase B was gradually increased from 40% (V/V) to 80% (V/V) over 25 min. The spectra were recorded by a DAD detector at 380 nm. The flow velocity was set to 1 mL/min, and the injection volume was 10 μL.
Separation and identification of metabolites
A 5,000 mL scale-up of P. igniarius was cultured according to the above-mentioned fermentation conditions. After the 0.1 mg/mL TL was added, the co-culture was grown for another 3 days. The filtrate of the fermentation broth was concentrated to 500 mL and partitioned with EtOAc (3 × 500 mL). The EtOAc extract was then evaporated under reduced pressure to yield 290 mg of residue, which was subjected to Sephadex LH-20 column chromatography, eluted with petrol-CHCl3-MeOH (5:4:1) to produce five fractions (A-E). Fraction B was purified through reverse-phase preparative HPLC using a mobile phase of MeOH-H2O (45:55) to afford compound A (25.5 mg). Fraction D was separated by preparative RP-HPLC using MeOH-H2O (62:38) to afford compound B (7.0 mg). The structures of the target compounds were identified by 1D and 2D NMR as well as HRESIMS analysis. 1D- and 2D-NMR spectra were obtained at 400 MHz for 1H and 100 MHz for 13C, respectively, on Bluker 400 MHz spectrometers in methanol-d4 with solvent peaks used as references. HRESIMS data were measured using an Agilent 1290 Infinity II Accurate Mass Q-ToF LC/MS spectrometer.
Determination of HIP yield by external standard method
The concentrations of HIP and TL were determined by HPLC with an external standard at the wavelength of 254 nm, and a 20-80% methanol gradient was used for elution. the concentration points for the HIP standard curve were 1.0, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL, and the concentrations for the TL standard curve were 8, 4, 2, 1, 0.5, and 0.25 mg/mL. The linear standard equation was obtained by using the linear least square method. As in the previous methods description, P. igniarius was cultured in MMM liquid medium for 15 days and then fed TL at a final concentration of 0.1 mg/mL. Fermentation broth samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 32 h, 64 h, and 128 h after the addition of TL into the fermentation system, and then the samples were extracted with ethyl acetate and analyzed by HPLC.
Protein extraction and enzymatic hydrolysis
A 5 mm magnetic bead and 25 μL of lysis buffer were added to 5 mg mycelium samples. The final concentrations of PMSF and EDTA were 1 mM and 2 mM, respectively. The mycelium samples were allowed to rest for 5 minutes after the eddy oscillation, and the final concentration of DTT was 10 mM. The supernatant was collected by centrifugation at 4 ℃ for 20 min after 2 min oscillation with a tissue abrasive apparatus. The supernatant was treated with 10 mM DTT for 1 hour in a water bath at 56 ℃. After coming back down to room temperature, IAM was added at a final concentration of 55 mM, and then the sample was kept in the dark for 45 minutes. The supernatant was precooled with 5× volume of acetone, precipitated at -20 ℃ for 2 h, and was centrifuged at 9000 × g, 4 ℃ for 20 min. The addition of acetone was repeated three times, followed each time by centrifugation and discarding the supernatant until the supernatant was colorless. Then, 25 μL of lysis buffer was added into the precipitation mixture. After 5 minutes of ultrasonication in an ice bath, the supernatant was centrifuged at 9000 × g, 4 ℃ for 20 min.
iTRAQ marker and peptide segment separation
First, 100 μg protein solution was extracted from each sample, and the trypsin enzyme was added in the ratio of protein:enzyme of 40:1. The enzyme was hydrolyzed for 4 hours at 37 ℃, at which point trypsin was again added (the same amount), and the mixture was continuously hydrolyzed for another 8 hours. The resulting enzymatically generated peptides were desalted using a Strata X column and then vacuum-dried.
Eight groups of iTRAQ labeling reagents (113, 114, 115, 116, 117, 118, 119, 121) were selected. Then, 50 μL isopropanol were added to each tube at 25 ℃, and the mixture was centrifuged after swirl oscillation. The supernatants were transferred to another clean sample tube along with the enzymatic peptides in 0.5 M TEAB. The different peptide fragments were labeled with their respective peptide’s labels.
The above samples were separated by liquid phase chromatography with a LC-20AB system (Shimadzu, Japan), and the separation column used was a Gemini C18 column (5 μm, 4.6 × 250 mm). The above dried samples were re-dissolved with phase A (5% ACN, pH 9.8), and the flow rate gradient was set as: 5% mobile phase B (95% ACN, pH 9.8) for 10 minutes, 5-35% mobile phase B for 40 minutes, 35-95% mobile phase B for 3 minutes, and 5% mobile phase B for 10 minutes, at rate of 1.0×10-3 L/min. The detection wavelength used was 214 nm. The fractions were collected once per minute, and the sample components were merged by chromatographic elution peaks to yield a total of 20 fractions. Those 20 fractions were frozen and concentrated each to the same volume of 1.0 mL.
LC-MS/MS analysis
After centrifugation for 10 minutes, the supernatant samples were separated using LC-20AD nanoflow liquid chromatography. A trap column was used to concentrate protein and remove salts. It was connected with a self-assembled C18 column (75 micron inner diameter, 3.6 micron column diameter, 15 cm column length) in series. The flow rate was set to 300 nL/min. Separation was carried out using the following gradients:
(1) 0-8 minutes, 5% mobile phase B (98% acetonitrile aqueous solution containing 0.1% formic acid (FA));
(2) 8-43 minutes, 8-35% mobile phase B gradient;
(3) 43-48 minutes, 35-60% mobile phase B gradient;
(4) 48-50 minutes, 60-80% mobile phase B gradient;
(5) 50-55 minutes, equivalent 80% mobile phase B;
(6) 55-65 minutes, equivalent 5% mobile phase B.
After ionization from a nano ESI source, the peptide segments were analyzed by high resolution liquid chromatography-mass spectrometry (HR-LC-MS) TripleTOF 5600. Using Proteome Discoverer, a Thermo Scientific tool, the original mass spectrometry file was converted into a MGF format file containing the information of secondary mass spectrometry (MS/MS) spectra, in which "BEGIN IONS" and "END IONS" were the starting and ending positions of each spectrum. The UniProt protein database was used to identify the proteins. The MGF file and protein database were searched to obtain the final protein identification results using the Mascot 2.3.02 identification software.
iTRAQ data analysis
iTRAQ data were quantified by IQuant software. The spectra and the list of peptide segments were filtered by 1% FDR (PSM-level FDR < 0.01) to identify significant results. According to the parsimony principle, the peptide segments were used to assemble proteins and produce a series of proteomes. In order to control the false positive rate of proteins, the process was again filtered at the protein level with 1% FDR (Protein-level FDR < 0.01). Quant's workflow included the following steps: protein filtering, purity correction of report group labels, normalization of quantitative values, complementation of missing values, calculation of quantitative values of proteins, statistical analysis, and display of final results.
Bioinformatic analysis
In this study, three sets of experiments including Control, TLPF32h, TLPF128h, and three sets of comparisons of TLPF32h /control, TLPF128h/control, TLPF128h/TLPF32h were conducted. The differentially expressed proteins (DEPs) were screened on the basis of a fold change > 1.5 and P < 0.05.
In the GO enrichment analysis of DEPs, the GO entries with significant enrichment were identified by hypergeometric test, compared with all identified proteins as background. The principle of Pathway enrichment analysis was similar. The hypergeometric test formula was as follows:
N: The number of GO entries matched in all identified proteins; n: the number of GO entries matched in DEPs; M: one of the GO entries matched in all identified proteins; m: one of the GO entries matched in DEPs. DEPs were determined to be significantly enriched in the GO entry if the P value of the hypergeometric test was less than 0.05.
Validation of MRM Technology
According to the results of iTRAQ data, differentially expressed proteins were screened and selected for MRM validation. Each sample was treated with 100 μg protein solution. Trypsin enzyme was added at a ratio of protein:enzyme of 40:1, with a total amount of 2.5 μg of trypsin enzyme, and enzymatic hydrolysis was carried out at 37 ℃ for 4 hours. Trypsin was added one more time at the same ratio, and the enzymatic hydrolysis was continued at 37 ℃ for another 8 hours. The enzymatic peptides were desalted using a Strata X column and then vacuum-dried.
The samples were scanned with a HPLC-TripleTOF 5600 mass spectrometer, and the resulting data were searched by Mascot v2.3 using the Fungi protein database (11,243 sequences) added to the internal standard peptide sequence. The DAT files were imported into Skyline software to establish the atlas library (credibility > 0.95). Skyline software was used to select target peptide segments under the following conditions:
(1) The peptide segments had matched second-order ions.
(2) The length of the peptide segments were between 5-40 amino acids.
(3) The peptide segment was the only one for the target protein.
(4) The cysteine in the peptide segment was modified to carbamidomethyl.
(5) There was no variable modification of the peptide segment.
(6) No methionine was found in the peptide segment.
(7) There was no missing cut of the peptide segment.
Transition selection set was determined as follows:
(1) The fragment ions were B and Y ions;
(2) The parent ion charges were 2,3,4;
(3) The charges of fragment ions were 1,2;
(4) Debris ion charge-mass ratio < 1,250 (four-stage rod scanning range);
(5) The number of transitions was 6.
Validation of MRM method
Skyline set up the MRM method and output it to the QTRAP 5500 mass spectrometer for MRM scanning verification. The success of MRM mass spectrometry for target proteins depended on the validation results, which must conform to:
(1) There were co-elution peaks in transitions with different peptide segments.
(2) The chromatographic peak area intensity of transitions with different peptide segments was correlated with the fragmentation intensity of spectral data.
(3) The retention time of the peptide segments in MR and full-spectrum scans was good.
MRM Mass Spectrometry Detection
The peptide fragments were separated by liquid phase and entered into the QTRAP 5500 tandem mass spectrometer. The ion source was Nanospray Illsource. In data acquisition, the instrument parameters are set as follows: spray voltage was 2,400V, spray gas was 23. With MRM scanning mode, the resolution of Q1 and Q3 was set to Unit mode.
Data analysis
Each transition signal of the target protein was normalized to the signal of beta-galactosidase. After normalized intensity, a linear mixed model integrated with the MS stats tool was used to quantify the target protein in the sample. This model gave the ratio of protein in the comparison group and the adjusted p-value. The corrected P value reflected the false positive rate of the original statistical test (Benjamin and Hochberg). If the difference in final target protein concentration was at least 1.5-fold with P < 0.05 (false positive < 0.05), the protein was considered to be significantly differentially expressed.